| ZHU Yanbing,GAO He,LI Hebin,NI Hui,JIANG Zedong,LI Lijun,XIAO Anfeng. 2019. Overexpression and characterization of a thermostable β-agarase producing neoagarotetraose from a marine isolate Microbulbifer sp. AG1. Acta Oceanologica Sinica, 38(2):96－106 |
| Overexpression and characterization of a thermostable β-agarase producing neoagarotetraose from a marine isolate Microbulbifer sp. AG1 |
| 产新琼四糖的海洋微泡菌属AG1热稳定性β-琼胶酶的过量表达与鉴定 |
| Received:January 14, 2017 |
| DOI：10.1007/s13131-019-1349-y |
| Key words:thermostable β-agarase neoagarotetraose Microbulbifer sp. |
| 中文关键词: 热稳定 β-琼胶酶 新琼四糖 微泡菌属 |
| 基金项目:The Natural Science Foundation of Fujian Province of China under contract No. 2016J01162; the Program for New Century Excellent Talents in Fujian Province University, China under contract No. B15139. |
| Author Name | Affiliation | E-mail | | ZHU Yanbing | College of Food and Biological Engineering, Jimei University, Xiamen 361021, China
Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China
Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China
Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China | | | GAO He | College of Food and Biological Engineering, Jimei University, Xiamen 361021, China | | | LI Hebin | Department of Pharmacy, Xiamen Medical College, Xiamen 361023, China | | | NI Hui | College of Food and Biological Engineering, Jimei University, Xiamen 361021, China
Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China
Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China
Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China | | | JIANG Zedong | College of Food and Biological Engineering, Jimei University, Xiamen 361021, China
Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China
Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China
Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China | | | LI Lijun | College of Food and Biological Engineering, Jimei University, Xiamen 361021, China
Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China
Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China
Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China | | | XIAO Anfeng | College of Food and Biological Engineering, Jimei University, Xiamen 361021, China
Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China
Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China
Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China | xxaaffeng@jmu.edu.cn |
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| Abstract: |
| An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21 (DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively. Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-D-galactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities. |
| 中文摘要: |
| 从微泡菌属AG1（Microbulbifer sp. AG1）克隆得到1302 bp大小的琼胶酶基因，该基因编码产物为一个成熟蛋白（413个氨基酸残基）外加一个信号肽（20个残基）。将不含信号肽片段的琼胶酶在E. coli BL21 （DE3）中进行了异源表达和纯化。使用琼脂糖作为底物，该重组琼胶酶的最适反应温度和pH分别为60℃和7.5。该重组酶表现出优良的热稳定性，在50℃和60℃下处理1 h，重组琼胶酶仍能分别保持67%和19%的残余酶活力。除了SDS，重组琼胶酶对于其他测试的抑制剂、去垢剂和尿素变性剂有着较好的抗性。利用薄层色谱和以对硝基苯-α/β-D-吡喃半乳糖苷为底物的酶活力分析结果表明，该重组琼胶酶为β型琼胶酶，它水解琼脂糖的主要终产物为新琼四糖，而且不同聚合度的酶解产物具有抗氧化活性。 |
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